By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to small-molecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule focused process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, many proteins are not amenable to such extensive mutagenesis. Relatively little is known about how to dramatically resurface a protein with a polycationic feature in a manner that does not dramatically alter or abolish its utility and/or function (stability, target affinity, expression in E. coli). Even structurally similar proteins respond differently to such extensive mutagenesis, and many proteins of therapeutic interest were not amenable to polycationic resurfacing.
Thus, there is a need in the art for the development of a protein scaffold that is amenable to cationic resurfacing and can penetrate the cell while also being able to recognize a magnitude of intracellular targets. Such a protein would represent a general scaffold for intracellular targeted protein therapeutic discovery.